**2. General**

Biophotolysis of water, fermentation and photofermentation of organic substrates are considered to be the best biological methods of hydrogen generation. Reversibility, lack of toxic substances generated in these processes, mild conditions for microbiological reactions, as well as operation at low pressure of these processes are the conditions required for

Microbiological Methods of Hydrogen Generation 225

they absorb light from the visible part of the light spectrum in which bacteriochlorophyll is not active and protect the antenna system against damage by singlet oxygen (Isaacs, 1995, Jones, 1997). The majority of the purple bacteria have two different antenna complexes known as LH1 and LH2. The number of LH2 complexes depends on such parameters like light intensity and partial pressure of oxygen, while the number of LH1 complexes is directly correlated with that of the reaction center (RC) to form RC-LH1 center. High ratio of pigment molecules to RC (e.g. 100 molecules of chlorophyll to one RC) increases the area capable of light absorption. Upon absorption of photon by LHC, the reaction centre becomes excited with simultaneous charge separation in a time shorter than 100 picoseconds (ps). The high reaction rate of this process is a consequence of the mutual arrangement of LH1 and RC: one RC is surrounded by a ring of 15-17 LH1 subunits. The closed structure of LH1 complexes in combination with the dense packing of bacteriochlorophyll molecules ensures fast delocalization of the excited state and possibility of energy transfer towards the reaction centre from every point of the ring (Vermeglio, 1999). The reaction centre is an integral part of protein membrane composed of three polypeptides (subunits L, M and H), containing four molecules of bacteriochlorophyll *a* (PA, PB, BA, BB), two molecules of bacteriofeophityne *a* (HA, HB), two molecules of ubichinone(QA, QB), one molecule of carotenoid (Crt) and one

Fig. 2. Reaction center (RC) of photosystem in *Rhodobacter sphaeroides* bacteria (Isaacs, 1995)

All pigments are linked to the heterodimeric protein skeleton of L and M subunits forming five transmembrane protein helixes (Paschenkoa, 2003). The main source of electrons is the "special pair" of the excited bacteriochlorophylls *a* located close to side of the cytoplasmic membrane. The excitation is realized by direct absorption of light by the "special pair" of bacteriochlorophylls absorbing at 870 nm and by energy transfer from other pigment

atom of non-heme iron (Fig.2).

modern microbiological systems. Moreover, the possibility of application of different waste waters (containing organic carbon) in these processes is an additional benefit.

Fermentation is the process generating basically two gaseous metabolites: hydrogen and carbon dioxide. The volatile fatty acids (VFA) and alcohols represent liquid metabolites of dark fermentation. The low yield of generated hydrogen and high concentration of CO2 (almost 50%) in gaseous products are the main disadvantages of microbiological hydrogen generation. In contrary, high reaction rate and possibility of biodegradation of many organic substances can be assigned to the benefits of this process.

In photofermentation, the photosynthetic heterotrophoic bacteria under anaerobic conditions and in the absence of nitrogen generate hydrogen in presence of organic compounds. Nitrogenase is the enzyme catalyzing hydrogen generation reaction. Presence of molecular nitrogen or nitrogen compounds directs the reaction route towards ammonia formation. The possibility of application of wide spectrum of light (400-950 nm), lack of methabolism generating molecular oxygen, as well as possibility of use of organic substances originating from wastes are the main advantages of photobiological method of hydrogen generation.

Both fermentation and photofermentation require presence of anaerobic microorganisms and the light in case of photofermentation. Photosynthesis, and in consequence also photofermentation is the series of complex reactions transforming energy of light into chemical energy.

Fig. 1. Scheme of photoinduced cyclic flow of electrons in photosystem of *Rhodobacter sphaeroides* bacteria (Vermeglio, 1999).

The photosynthetic apparatus is localized in invaginations of the cytoplasmic membrane. The photosystem is built of three multimeric (transmembrane) proteins: antennas making the light-harvesting complex (LHC), the reaction centre (RC) and the complex of cytochromes *bc1* (Fig.1) (Vermeglio, 1999). The LHC antennas contain molecules of bacteriochlorophyll and carotenoides. The carotenoides play a double role in LHC systems;

modern microbiological systems. Moreover, the possibility of application of different waste

Fermentation is the process generating basically two gaseous metabolites: hydrogen and carbon dioxide. The volatile fatty acids (VFA) and alcohols represent liquid metabolites of dark fermentation. The low yield of generated hydrogen and high concentration of CO2 (almost 50%) in gaseous products are the main disadvantages of microbiological hydrogen generation. In contrary, high reaction rate and possibility of biodegradation of many organic

In photofermentation, the photosynthetic heterotrophoic bacteria under anaerobic conditions and in the absence of nitrogen generate hydrogen in presence of organic compounds. Nitrogenase is the enzyme catalyzing hydrogen generation reaction. Presence of molecular nitrogen or nitrogen compounds directs the reaction route towards ammonia formation. The possibility of application of wide spectrum of light (400-950 nm), lack of methabolism generating molecular oxygen, as well as possibility of use of organic substances originating from wastes are the main advantages of photobiological method of

Both fermentation and photofermentation require presence of anaerobic microorganisms and the light in case of photofermentation. Photosynthesis, and in consequence also photofermentation is the series of complex reactions transforming energy of light into

Fig. 1. Scheme of photoinduced cyclic flow of electrons in photosystem of *Rhodobacter* 

The photosynthetic apparatus is localized in invaginations of the cytoplasmic membrane. The photosystem is built of three multimeric (transmembrane) proteins: antennas making the light-harvesting complex (LHC), the reaction centre (RC) and the complex of cytochromes *bc1* (Fig.1) (Vermeglio, 1999). The LHC antennas contain molecules of bacteriochlorophyll and carotenoides. The carotenoides play a double role in LHC systems;

waters (containing organic carbon) in these processes is an additional benefit.

substances can be assigned to the benefits of this process.

hydrogen generation.

chemical energy.

*sphaeroides* bacteria (Vermeglio, 1999).

they absorb light from the visible part of the light spectrum in which bacteriochlorophyll is not active and protect the antenna system against damage by singlet oxygen (Isaacs, 1995, Jones, 1997). The majority of the purple bacteria have two different antenna complexes known as LH1 and LH2. The number of LH2 complexes depends on such parameters like light intensity and partial pressure of oxygen, while the number of LH1 complexes is directly correlated with that of the reaction center (RC) to form RC-LH1 center. High ratio of pigment molecules to RC (e.g. 100 molecules of chlorophyll to one RC) increases the area capable of light absorption. Upon absorption of photon by LHC, the reaction centre becomes excited with simultaneous charge separation in a time shorter than 100 picoseconds (ps). The high reaction rate of this process is a consequence of the mutual arrangement of LH1 and RC: one RC is surrounded by a ring of 15-17 LH1 subunits. The closed structure of LH1 complexes in combination with the dense packing of bacteriochlorophyll molecules ensures fast delocalization of the excited state and possibility of energy transfer towards the reaction centre from every point of the ring (Vermeglio, 1999). The reaction centre is an integral part of protein membrane composed of three polypeptides (subunits L, M and H), containing four molecules of bacteriochlorophyll *a* (PA, PB, BA, BB), two molecules of bacteriofeophityne *a* (HA, HB), two molecules of ubichinone(QA, QB), one molecule of carotenoid (Crt) and one atom of non-heme iron (Fig.2).

Fig. 2. Reaction center (RC) of photosystem in *Rhodobacter sphaeroides* bacteria (Isaacs, 1995)

All pigments are linked to the heterodimeric protein skeleton of L and M subunits forming five transmembrane protein helixes (Paschenkoa, 2003). The main source of electrons is the "special pair" of the excited bacteriochlorophylls *a* located close to side of the cytoplasmic membrane. The excitation is realized by direct absorption of light by the "special pair" of bacteriochlorophylls absorbing at 870 nm and by energy transfer from other pigment

Microbiological Methods of Hydrogen Generation 227

The second group of fermentative bacteria active in hydrogen generation belongs to anaerobic gram-negative bacteria. The best activity in biohydrogen generation *via* dark fermentation was found for the following strains: *Enterobacter asburiae* (Jong-Hwan, 2007)*, Enterobacter cloacae* (Mandal, 2006)*, Enterobacter aerogenes* (Jo, 2008)*, Escherichia coli* (Turcot, 2008)*, Klebsiella oxytoca* (Wu, 2010) or *Citrobacter Y19* (Oha, 2003). These strains of bacteria can tolerate oxygen in environment. Here, in aerobic condition the oxygen respiration can occurs. The change of metabolic pathway provides method for survival under variable conditions of environment. These bacteria show better biological activity in comparison with those active only in completely anaerobic conditions. However, in aerobic conditions no hydrogen formation is observed. This effect is caused by inhibition of hydrogenase,

Thermophilic bacteria operating at 60-85 oC belongs to the third group of bacteria generating hydrogen in fermentative processes (Zhang, 2003). The following strains of thermophilic bacteria of *Thermoanaerobacterium thermosaccharolyticum* (Thonga, 2008) and hyperthermophilic of *Thermatoga neapolitana* (Mars, 2010, Eriksen, 2008), *Thermococcus kodakaraensis* (Kanai, 2005)*,* or *Clostridium thermocellum* (Lewin, 2006) can generate hydrogen in presence of organic substrates at relatively high temperatures. It was established that

Application of *C. saccharolyticus* and *Thermatoga elfii* thermophilic bacteria results in 80 % yield of the theoretical one (theoretically 4 moles of glucose can be transformed into acetic acid with 100% yield) while applying saccharose or glucose (Vardar-Schara,2008), respectively. High yield in hydrogen generation is explained by Guo *et al.* (Guo, 2010) who assumes that high temperature can accelerate hydrolysis of substrates engaged in this process. At the same time Valdes-Vazquez et al. (Valdes-Vazquez, 2005) demonstrates that such results are not surprising, because optimal activity of hydrogenase is 50-70 °C. Unfortunately, the high yield of hydrogen generation with thermophilic bacteria is not equivalent to total amount of generated gas (Hallenbeck, 2009). In this situation the construction of bigger reactors is required what in consequence increase total costs. Moreover, reaction performed at higher temperatures require additional thermal energy

Photofermentation in hydrogen generation is the process which requires appropriate strain of bacteria, organic substances (mainly VFA) and light with appropriate intensity. The following strains of bacteria indicate activity in photoproduction of hydrogen: *Rhodobacter sphaeroides* (Koku, 2002)*, Rhodobacter capsulatus* (Obeid, 2009), *Rhodovulum sulfidophilum*  (Maeda, 2003)*, or Rhodopseudomonas palustris* (Chen, 2008). The research of new strains active in photogeneration of hydrogen is performed in numerous laboratories all over the world. These efforts were recently awarded by discovery of activity in *Rheudopseudomanas faecalis*

*Rhodobacter sphaeroides* belong to the group of bacteria the best recognized in hydrogen generation. These gram-negative bacteria belongs to the purple non-sulfur (PNS) *Proteobacteria* subgroup (Porter, 2008). The morphology is different because the shape of these bacteria as well as their dimensions strongly depends on the medium (see Fig. 3). In medium containing sugars the dimensions are limited to 2.0-2.5 x 2.5-3.0 μm, whereas under

other conditions they can vary from 0.7 to 4.0 μm (Garrity, 2005).

thermophilic bacteria are the most effective from all those already described.

enzyme catalyzing hydrogen generation.

supplied to the bioreactor.

(Ren, 2009).

molecules located at RC or LHC. The transfer of electrons from the special pair to bacteriopheophytin, located in the middle of the dielectric cytoplasmic membrane occurs in 3-4 ps. This reaction is probably intermediated by a transient product of monomeric bacteriochlorophyll BA. In the next 200 ps the electron is transferred to ubiquinone QA (connected with RC) and subsequently to ubiquinone QB. The transfer of electron to ubiquinone QB is accompanied by its protonation. The full reduction of ubiquinone QB requires two subsequent cycles in RC after which electrons finally leave RC with electrostatically neutral doubly reduced ubiquinol QH2 (Jones, 1997). The two protons required for protonation originate from cytoplasmic space. In the next step ubiquinol is oxidized by the *bc1* cytochrome complex. This complex caused reduction of the [Fe2S2] unit which is a part of cytochrome (part of Rieske unit) and releases two protons to periplasmic space. Then the cycle of electron transfer is closed by recombination of cytochrome *c2* by reduction of the special pair of bacteriochlorophylls. The cyclic transfer of electrons is accompanied by transfer of protons from cytoplasm to periplasm leading to the proton gradient between the two sides of cytoplasmic membrane, which is the most important effect of photosynthesis because it stimulates ATP synthesis and reduction of NAD+ (Vermeglio, 1999). Protons accumulated on the periplasmic space of the membrane return to the cytoplasmic space through the ATP synthase channel, which closes the transfer of protons (Paschenkoa, 2003).
